Physical properties of apolipoprotein A-I from the chicken, Gallus domesticus

The amphipathic alpha-helices of exchangeable apolipoproteins (apo) function to simultaneously facilitate interaction with lipid surfaces and the aqueous environment. In contrast to mammalian apoA-I's, which self-associate in the absence of lipid, chicken apoA-I, which shares 66% sequence homology with human apoA-I, exists as a monomeric protein when dissociated from high-density lipoprotein (HDL). Sedimentation equilibrium studies conducted in the analytical ultracentrifuge yielded a weight-average molecular weight of 28 170. Corresponding sedimentation velocity and diffusion experiments gave rise to s(0)20,w = 2.23 S and D(0)20,w = 6.39 x 10(-7) CM(2)/S. A translational frictional ratio (f/fmin.) of 1.18 and an axial ratio of 4.0 were also determined from this data. The Stokes radius (Rs,sed = 2.80 nm) and translational frictional ratio were subsequently used to calculate estimated molecular dimensions of 25.2 X 100.8 angstroms for chicken apoA-I. Circular dichroism (CD) studies revealed a highly alpha-helical structure predicted to be 74% by Provencher-Glockner analysis. Denaturation studies performed on lipid-free apoA-I and monitored by CD revealed a midpoint of denaturation of 0.64 M guanidine hydrochloride. From plots of deltaGapp versus guanidine hydrochloride concentration, a deltaGD(H2O) of 1.86 kcal/mol was determined. In other studies, a midpoint of temperature-induced denaturation for apoA-I of 57 degrees C was obtained. The effect of solvent pH on the secondary structure content of apoA-I revealed a significant loss of alpha-helix below pH 4.0 and above pH 10, suggesting that lipid-free apoA-I may by partially stabilized by the formation of intra- or interhelix salt bridges between oppositely charged amino acid side chains. Denaturation studies of apoA-I bound to the surface of HDL revealed a midpoint of guanidine hydrochloride induced denaturation of 3.25 M, indicating that considerable stability is conferred on this